Summary Infants have a high incidence of severe infections and low immune responses to vaccines. HIV-exposed uninfected infants (HEUs) have even higher incidence of severe infections, hospitalizations and death than HIV-unexposed uninfected infants (HUUs) and lower responses to vaccines. We hypothesized that the global immune dysfunction of HEUs results from excessive Tregs that develop in response to 1) a highly inflammatory in utero environment of HIV-infected pregnant women and 2) infant gut dysbiosis. These hypotheses will be addressed in the following Specific Aims: AIM 1. To measure the proportions of Tregs in HEUs and HUUs during the 1st year of life and to identify correlations between the abundance of Tregs and decreased immune responses to live vaccines. This will be accomplished by comparing the Treg subsets at birth, 6 months and 1 year of life of 100 HEUs and 100 HUUs from Soweto, South Africa, and by correlating the frequency of dominant Treg subsets (measured by flow cytometry) with the cellular immune responses to BCG vaccine (measured by ELISPOT) and with their antibody responses to measles vaccine (measured by ELISA). Both vaccines are live attenuated and therefore generate immune responses that mimic the responses to wild type infections. AIM 2. To identify differences in the composition and diversity of the gut microbiota between HEUs and HUUs, which are associated with Treg subsets and their responses to vaccines. The gut microbiota have a prominent and well-established effect on the development of the immune system and on Tregs in particular. We will compare the microbiomes of HEUs and HUUs by high throughput sequencing of 16S rRNA and will investigate correlations with Treg subsets and immune responses to vaccines. AIM 3. To compare the HEU and HUU epigenetic profiles critical for Treg differentiation and function. Taking advantage that there are known key sites whose methylation status determines Treg differentiation, we will compare these regions in HEUs and HUUs. The completion of these Aims will open the possibility of indirectly educating the neonate immune system by modulating the infant and/or maternal gut microbiomes and/or by designing vaccines resistant to Tregs.